Abstract-
Bacteria are the natural world 's unsung heroes. They receive bad rep due to the disease causing individuals like tuberculosis but the vast majority of bacteria on Earth are harmless if not beneficial to both the environment and humans. Take for example E. Coli a well known bacteria that lives within our intestines. This particular bacteria makes vitamins that we need in order to stay healthy. In this experiment we analyzed the changes seen in bacteria when adding to differing DNA plasmids; pUC18 and lux. To arrive to the most accurate results possible we had to have the E. Coli bacteria made permeable to the plasmids. So it was prepared with calcium chloride. One group of bacteria was given the pUC18 plasmid, and the other group was given the lux as well as the pUC18 plasmids. We then proceeded to incubate them in different containers. Some had ampicillin where as some had none. This was done in order to observe the effect of pUC18, and lux growth patterns. A control with neither plasmid was used to keep a basis for our experiments. Bacteria that had transformed and taken in the lux plasmid without ampicilin were able to illuminate, and only bacteria that gained the pUC18 were capable of surviving with ampicillin. The results observed reveal how foreign DNA can be adapted to fit into another cell 's DNA even incorporated to be an optimal part of the cell. This process makes it a very viable manner of being able to mass replicate advantageous genes, that would
This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.
As predicted the E. coli colony transformed with either the PUC18 or the lux plasmid developed an ampicillin resistance. Which made it easier for them to not only survive but also replicate in both the LB agar plates and the LB ampicillin rich agar plate. However the E. coli colony not treated with the plasmids could not survive and colonize in the LB ampicillin rich agar plates. The plate that had no ampicillin in its environment and no plasmid treated E. coli served as a positive control for this experiment because it demonstrated how the E. coli would colonize and grow in a normal setting. The cells in the positive control plate grew into lawn colonies because they were not placed into a selective environment or transformed, so they had no need to acquire ampicillin resistance. Two plates in the experiment contained E. coli cells that were transformed with either the PUC18 or the lux plasmid but were placed in an ampicillin free environment. These two colonies grew
Control plasmids lux and pUC18 were introduced into E. Coli through a process of transformation.
The color of the bacteria was a whitish color and the colony size is similar both before and after the transformation. The best way to do it is to compare the control of the experimental plates. Cells that were typically not treated with the plasmid could not grow on ampicillin, although cells that were treated with the plasmid can grow on the LB/AMP plate. The plasmid would have to confer resistance to ampicillin. Moving on, the GFP gene is what is glowing in the plate because it was activated by the sugar arabinose. The sugar arabinose and the plasmid DNA are also needed to be present because that is what initially turns on the GFP gene which makes the bacteria glow. Organisms can also turn on and off particular genes for camouflage reasons. An organism would benefit from turning on and off certain
This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.
70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first
An unknown bacterium 15 was awarded and labeled at the table ready to be identified. Using the skills and test that are taught and learned in microbiology were applied into learning what the unknown bacteria culture was. There were multiple procedures and test done in order to gain all the information needed to determine which bacteria was given. In order to find what the bacteria was the first step was finding the right environment and temperature that would allow the bacteria to thrive and grow. Determining this is one of the most important steps in being able to obtain conclusive results that would allow the results of the test to be accurate and correct. Without the correct temperature and environment the bacteria will give inconclusive results which will alter and skew the end results and may lead to the wrong conclusion. By using the methods that were obtained and learned through the microbiology class allowed the skills and knowledge to determine the bacteria and execute the tests in order to determine the culture.
The purpose of this experiment is to make E.Coli competent so that it can be transformed in order to become immune to ampicillin, then we would be able to determine the transformation efficiency of the culture. We determine this by preparing 4 plates of E.coli, each labeled “LB-plasmid”, “LB+plasmid”, “LB?Amp-plasmid”, and “LB/Amp+plasmid”. This meant that either should have lacked plasmid and Ampicillin, with plasmid but lacked Ampicillin, without plasmid but with Ampicillin, or were with Ampicillin and plasmid, respectively. Then we made the bacterial cells competent by adding CaCl2 to 2 vials of the colony (one with plasmids), and incubating on ice, then heat shocking, and returning to ice. Luria Broth is then added and left to sit for 5-15
The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies
How does the addition of pGLO plasmid to a solution containing E. coli bacteria affect the growth and characteristics of the bacteria? Genetic transformation is the incorporation of foreign DNA into an organism to potentially change the organism’s trait. Plasmids are small circular DNA that replicate separately from the bacterial chromosome. In nature, these plasmids can be transferred between bacteria allowing for the sharing of beneficial genes. Due to this characteristic, plasmids allow for genetic manipulation and can be moved between bacteria easily. The pGLO plasmid utilized in this experiment encodes the gene for Green Fluorescent Protein (GFP), which under the right conditions can produce a glow. The gene regulation system present in the pGLO plasmid requires
The field of biotechnology involves the concept of genetic engineering, altering the DNA/genetic material of an organism using information from a different one. The process in which bacteria can obtain this manipulated genetic information from another source is called genetic transformation. The goal of this experiment was to genetically transform Escherichia coli bacteria’s DNA by inserting the vector pGLO plasmid which codes for ampicillin resistance as well as the green fluorescent protein, GFP. For the experiment, the E. coli bacteria were separated into two groups; control and
Coli. The first standard E. Coli has no resistance plasmid while the second strain contains a resistance plasmid with genes protecting it from ampicillin. This standard E. Coli and pAMP (plasmid-Ampicillin) E. Coli were each streaked across plates containing the antibiotic and containing growth supportive Lurithea Broth. The purpose of this lab was to test their growth in each medium. Our hypothesis was that while the ampicillin resistant E. Coli would show growth in both LB and LB-AMP plate, the standard E. Coli would only grow in the LB plate for it contains no resistant plasmids against the
It will also highlight how successfully integrated plasmids can affect the cell and what proteins are produced by the DNA, and how easy it is to change the genetics of cell and the future generations of that cell. The experiment also demonstrates how natural selection would work in a lab environment, and how the addition of a gene or a mutation can protect an organism from adverse conditions in the environment. This lab also illustrates the change in DNA in a visual sense. Molecular genetics is a very small scale science that cannot be seen by the human eye, however by illuminating the bacteria with the GFP it will produce the very small process can be clearly seen before and after the genetic splicing. The bacteria will produce quickly and the successful bacteria will be clearly seen by the human eye, even after generations upon generations of bacteria.
Scientists can study and manipulate genes by using precisely engineered plasmids, According to ADDGENE, a nonprofit plasmid repository, plasmids “have become possibly the most ubiquitous tools in the molecular biologist’s toolbox” (“What Is a Plasmid?”). In this experiment, we will use pGLO to genetically transform the bacteria E. coli. pGLO is a genetically engineered plasmid that carries the reporter genes for both green fluorescent proteins (GFP) and the ampicillin resistance. GFP, a protein typically found in the bioluminescent jellyfish Aequorea victoria, exhibits a bright green fluorescence in the presence of blue to UV wavelengths (Mecham); the protein absorbs UV light from the sun and emits it as a lower energy green light, exemplifying the second law of
Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial